Predicting mortality and detecting severe disease

ABSTRACT

Measurement of circulating ST2 and/or IL-33 concentrations is useful for the prognostic evaluation of subjects, in particular for the prediction of adverse clinical outcomes, e.g., mortality, and the detection of severe disease.

CLAIM OF PRIORITY

This application is a continuation application of U.S. patentapplication Ser. No. 13/282,111, filed on Oct. 26, 2011 (issued as U.S.Pat. No. 9,568,481), which is a continuation application of U.S. patentapplication Ser. No. 13/179,173, filed on Jul. 8, 2011 (issued as U.S.Pat. No. 8,617,825), which is a continuation application of U.S. patentapplication Ser. No. 11/789,169, filed on Apr. 24, 2007 (issued as U.S.Pat. No. 7,998,683), which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/794,354, filed on Apr. 24, 2006, U.S.Provisional Patent Application Ser. No. 60/800,362, filed on May 15,2006, and U.S. Provisional Patent Application Ser. No. 60/904,608, filedon Mar. 2, 2007. The entire contents of each of the foregoingapplications are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to methods for predicting mortality and detectingthe presence of severe disease by measuring circulating levels of ST2and/or IL-33, alone or in combination with other biomarkers.

BACKGROUND

Clinical evaluation of subjects, particularly those with non-specificsymptoms such as chest pain or discomfort, shortness of breath, nausea,vomiting, eructation, sweating, palpitations, lightheadedness, fatigue,or fainting, can present significant challenges, as the cause andseverity of the subject's condition may not always be apparent. Thedecision whether to treat a subject aggressively or conservatively, orto admit the subject as an inpatient or to send them home, may sometimesbe made solely on a physician's clinical assessment or “gut feeling” asto the individual's actual condition. Biomarkers that indicate asubject's likelihood of an adverse outcome, e.g., mortality, and/or thepresence of severe disease, would significantly enhance the physician'sability to make informed treatment decisions.

SUMMARY

The present invention is based, at least in part, on the discovery thatserum levels of the biomarker ST2 (Growth Stimulation-Expressed Gene 2,also known as Interleukin 1 Receptor Like 1 (IL1RL-1)) can be used topredict clinical outcome, e.g., death, within a specific time period,e.g., 30 days, 3 or 6 months, or a year or more, or to detect thepresence of severe disease, regardless of the underlying causes ofsymptoms or ultimate diagnosis. Changes in the level of ST2 over time,e.g., over a few days or more, can also be used to predict clinicaloutcome, e.g., in patients hospitalized after an acute event.

The methods described herein include measuring ST2 levels as well asmonitoring changes in ST2 levels over time (e.g., ratios) to providediagnostic and prognostic evaluation of patients, e.g., patients withnon-specific symptoms, e.g., acutely dyspneic patients and those withchest pain.

IL-33 has been identified as the ligand for ST2. Thus, the inventionincludes methods for evaluating patients by monitoring biomarker levelsof ST2 and/or IL-33 levels, as well as ST2/IL-33 complexes, and ratiosof ST2:IL-33 complexes to free ST2 and/or IL-33.

In addition, the methods can include using additional diagnosticmethods, including evaluating organ function and/or levels of adjunctbiomarkers such as troponin (Tn, e.g., TnI or TnT), brain natriureticpeptide (BNP), proBNP, NT-proBNP, atrial natriuretic peptide (ANP),NT-proANP, proANP, C-reactive peptide (CRP), Blood Urea Nitrogen (BUN),D-dimers (degradation products of cross-linked fibrin, whose levelbecomes elevated following clot formation), albumin, liver functionenzymes, measures of renal function (e.g., creatinine, creatinineclearance rate, or glomerular filtration rate) and/or bacterialendotoxin. In some embodiments, the methods include measuring ST2 and/ora change in ST2 levels over time in addition to BUN, NT-proBNP or BNP,and/or TnI.

Thus, in one aspect, the invention features methods for evaluating therisk of death or readmission within a specific time period, e.g., 30,60, 90, or 180 days (e.g., one, two, three, or six months), or one, two,or five years, for a subject. The methods include obtaining a sample,e.g., blood, serum, plasma, urine, or body tissue from the subject;determining a biomarker level of ST2 and/or IL-33 in the sample; andcomparing the biomarker level of ST2 and/or IL-33 in the sample to areference level of ST2 and/or IL-33. A comparison of the biomarker levelof ST2 and/or IL-33 in the sample versus the reference indicates thesubject's risk of death or readmission within the specific time period.In some embodiments, the specific time period is one year.

In some embodiments, the reference level represents a level in a subjector group of subjects who have a low risk of death within one year. Insome embodiments, e.g., wherein the biomarker level of ST2 is measuredusing an immunoassay, e.g., an enzyme-linked immunosorbent assay(ELISA), e.g., as described in Example 1, the reference level of ST2 isbetween about 0.2 and 0.3 ng/ml of serum, e.g., the level can be 0.20,0.23, 0.25, 0.27, or 0.29 ng/ml of serum, and a level in the sample thatis greater than or equal to the reference level indicates that thesubject has an elevated, i.e., statistically significantly elevated,risk of death within one year. If an analytical technique other than theELISA described in Example 1 is employed, the reference ST2 level may bedifferent than described herein. However, the specific numbers recitedherein should be construed to be equivalent to corresponding numbersgenerated using other analytical techniques. In some embodiments, theelevated risk of death is at least 20% higher, e.g., 30%, 40%, or 50%higher.

In another aspect, the invention features methods for determining theseverity of one or more diseases, e.g., the present severity ofdiseases, in a subject. The methods include obtaining a sample from thesubject; determining a biomarker level of ST2 and/or IL-33 in thesample; and comparing the biomarker level of ST2 and/or IL-33 in thesample to a reference level of ST2 and/or IL-33. The biomarker level ofST2 and/or IL-33 in the sample as compared to the reference indicateswhether the one or more diseases the subject has are severe, e.g.,life-threatening.

In a further aspect, the invention includes methods for monitoring asubject's condition, e.g., for deciding whether a subject has improved,e.g., improved sufficiently to be discharged from the hospital. Themethods include determining a first biomarker level of ST2 and/or IL-33in the subject, e.g., a baseline level; and determining at least onesubsequent biomarker level of ST2 and/or IL-33 in the subject, e.g., atreatment level. Then, the first level and the subsequent levels arecompared. If the biomarker level of ST2 and/or IL-33 decreasessufficiently, e.g., statistically significantly, or by at least 5%, 10%,15%, 20%, or more, from the first to the subsequent levels, then thesubject's condition is likely to be improving and, if either one or bothlevels are low enough, e.g., below a selected threshold, then thesubject can be discharged, e.g., for outpatient treatment.

In some embodiments, the methods include determining a level of ST2 thatindicates a subject's risk, and optionally selecting or modifying atreatment for the subject, based on a ratio of a first ST2 level, e.g.,a baseline level, to a second ST2 level, e.g., a level taken some timelater, e.g., one, two, three, four, or more days later. For example, ifthe second level of ST2 is more than a selected percentage of the firstlevel, then the subject has a high risk and should be treated moreaggressively; if the subject is already being treated, then the subjectis not responding favorably to the current treatment and a new treatmentshould be selected, i.e., an alternate treatment to which the patientmay respond more favorably. As one example, if the second level is about85% or more of the first level (i.e., has decreased by about 15% orless), then the subject is not improving and still has a high risk ofdeath.

In some embodiments, the level of ST2 in a subject is compared to areference level that represents a level in a subject who does not havesevere disease, e.g., has no disease or has no acute, severe disease,e.g., when measured using an ELISA, e.g., as described herein. Thereference level of ST2 can be between about 0.2 and 0.3 ng/ml, e.g., thelevel can be about 0.20, 0.23, 0.25, 0.27, or 0.29 ng/ml of serum orplasma (as noted above, the thresholds recited herein apply when usingan ELISA method as described herein; other corresponding thresholdnumbers can be considered as equivalent to these numbers when determinedusing a different method). A level in the sample that is greater than orequal to the reference level indicates that the subject has one or moresevere diseases, e.g., present diseases.

In some embodiments, the reference level represents a subject with acertain prognosis. For example, when the level of ST2 is measured usingan ELISA, e.g., as described herein in Example 1, the reference levelcan be used to determine prognosis as follows: an ST2 less than about0.2 or 0.3 ng/ml indicates that the subject has a good prognosis, e.g.,is likely to recover; an ST2 of from about 0.2 or 0.3 ng/ml to 0.7 ng/ml(or an equivalent thereof) indicates that the subject has a poorprognosis, e.g., is less likely to recover. Finally, an ST2 of greaterthan 0.7 ng/ml indicates a very poor prognosis, e.g., the subject is notlikely to recover. In this embodiment poor prognosis would indicate thatthe patient is at a high risk of death or developing more severe diseasewithin one year possibly requiring hospital admission. Very poorprognosis indicates that the patient has a high probability of death ordeveloping more severe disease within 90 days possibly requiringhospital admission. In one study patients with an ST2 level higher than0.7 ng/ml had a mortality rate of over 30%.

In some embodiments, the subject exhibits one or more non-specificsymptoms, e.g., chest pain or discomfort, shortness of breath (dyspnea),nausea, vomiting, eructation, sweating, palpitations, lightheadedness,fatigue, and fainting. In some embodiments, the symptom is dyspnea orchest pain.

In some embodiments, the subject does not have a cardiovasculardisorder. In various embodiments, the subject has a pulmonary disorder,e.g., acute infection (e.g., pneumonia), chronic obstructive pulmonarydisease (COPD), and pulmonary embolism.

In certain embodiments, the subject has a liver disorder, e.g., a liverdisorder associated with chemotherapy, alcohol toxicity, or drugtoxicity as determined by standard liver function laboratory tests.

In some embodiments, the methods further include determining the levelof an adjunct (non-ST2, non-IL-33) biomarker, e.g., Troponin, NT-proBNP,BNP, proBNP, NT-proANP, proANP, ANP, CRP, D-dimers, BUN, albumin, liverfunction enzymes, measures of renal function, e.g., creatinine,creatinine clearance rate, or glomerular filtration rate, and/orbacterial endotoxin, in the sample; and comparing the level of theadjunct biomarker in the sample to a reference level of the adjunctbiomarker. The level of the adjunct biomarker in the sample as comparedto the reference, in combination with the level of ST2 in the sample ascompared to an ST2 reference level, indicates whether the subject has anelevated risk of death within a specific time period, and/or has apresent severe disease. In some embodiments, the methods includedetermining a change in levels over time (e.g., a ratio) for the adjunctbiomarker, by comparing a first level, e.g., a baseline level, to asecond level, e.g., a level taken some time later, e.g., one, two,three, four, or more days later. In embodiments where a ratio of ST2 iscalculated, a ratio of an adjunct biomarker can also be calculated,e.g., based on the same time period as the ratio of ST2.

In some embodiments, the subject has a BMI of 25-29, a BMI of ≥30, orrenal insufficiency, e.g., the subject is selected on the basis thatthey have a BMI of 25-29, a BMI of ≥30, or renal insufficiency.

In some embodiments, the methods include determining a level of ST2 anda level of IL-33 in the sample; determining a ratio of ST2:IL-33 in thesample; and comparing the ratio of ST2:IL-33 to a reference ratio. Theratio of ST2:IL-33 in the sample as compared to the reference ratioindicates whether the subject has an elevated risk of death within aspecific time period, and/or has present severe disease.

In another aspect, the invention provides methods for evaluating therisk of death within a specific time period, e.g., 30, 60, 90, or 180days (6 months), or one, two, or five years, e.g., for a subject whoexhibits one or more non-specific symptoms. The methods includeobtaining a sample from the subject; determining a biomarker level ofST2 and/or IL-33 in the sample, and optionally a level of NT-proBNP,proBNP, or BNP in the sample; and comparing the level of ST2 and/orIL-33 in the sample, and the level of NT-proBNP in the sample (ifdetermined), to corresponding reference levels. The level of ST2 and/orIL-33 in the sample, and the level of NT-proBNP, proBNP, or BNP in thesample, as compared to the respective reference levels indicate thesubject's risk of death within the specific time period.

In some embodiments, the methods include determining levels of (i)NT-proBNP, proBNP, or BNP and (ii) ST2 in the sample. In someembodiments, the subject's risk of death within one year is as follows:

ST2 < 0.20 ng/ml ST2 ≥ 0.20 ng/ml NT-proBNP < 986 pg/ml Lowest RiskMedium Risk NT-proBNP ≥ 986 pg/ml Medium Risk Highest Risk

In certain embodiments, if the subject has the highest level of risk ofdeath or hospital readmission, the subject is treated aggressively.

In a further aspect, the invention includes methods for monitoring asubject's condition, e.g., for deciding whether a subject has improved,e.g., improved sufficiently to discharge the subject from the hospital.The methods include determining a first level of (i) a non-ST2biomarker, e.g., NT-proBNP, proBNP, or BNP and (ii) ST2 and/or IL-33 inthe subject, e.g., a baseline level; and determining at least onesubsequent level of (i) the non-ST2 biomarker, e.g., NT-proBNP, proBNP,or BNP and (ii) ST2 and/or IL-33 in the subject, e.g., a treatmentlevel. Then, the first level and the subsequent levels are compared. Ifthe level of ST2 and/or IL-33 decreases from the first to the subsequentlevels, then the subject is likely to be improving and, if the level islow enough, then the subject has improved, e.g., improved sufficientlyto be discharged, e.g., for outpatient treatment. In some embodiments,the methods include determining at least a first, second, and thirdlevel of (i) a non-ST2 biomarker, e.g., NT-proBNP, proBNP, or BNP and(ii) ST2 and/or IL-33 in the subject, and comparing the levels. Adifference between the levels indicates whether the subject has improvedsufficiently to be discharged. Thus, for example, a decision todischarge or continue to treat on an inpatient basis can be made asfollows:

ST2 > threshold ST2 < threshold BNP > threshold Prognosis very poor,Patient may still need requires intensive treatment, favorabletreatment, do not discharge prognosis, possibly discharge as outpatientBNP < threshold Prognosis poor, patient Prognosis good may havecomplicating illness, if discharge monitor closely

In some embodiments, the threshold for ST2 is 0.2 ng/ml, and thethreshold for BNP is 986 pg/ml.

Also provided herein are kits including one or more antibodies that bindspecifically to ST2 and/or one or more antibodies that bind specificallyto IL-33, and instructions for performing one or more of the methodsdescribed herein.

In a further aspect, the invention provides methods for evaluating asubject's condition. The methods include obtaining a sample from thesubject; determining a biomarker level of ST2 and/or IL-33 in thesample, and determining presence or a level of one or more, e.g., all,of the following other biomarkers:

-   -   (i) NT-proBNP, proBNP, or BNP; (ii) NT-proANP, proANP, or        ANP; (iii) cardiac troponin (cTn), e.g., cTnl; (iv)        D-dimers; (v) C-reactive protein (CRP); (vi) creatinine,        creatinine clearance rate, or glomerular filtration rate; (vii)        Blood Urea Nitrogen (BUN); (viii) bacterial endotoxin; (ix) one        or more liver function enzymes; and        comparing the level of ST2 and/or IL-33 in the sample, and the        level of the one or more other biomarkers in the sample, to        corresponding reference levels. The level of ST2 and/or IL-33 in        the sample, and the level of the other biomarker in the sample,        as compared to the reference levels indicate the severity of the        subject's condition.

In some embodiments, the methods described herein include measuringlevels or ratios of ST2 and/or IL-33 in combination with BNP orNT-proBNP; with troponin, e.g., TnI or TnT; or with a measure of renalfunction, e.g., creatinine, creatinine clearance rate, or glomerularfiltration rate.

In another aspect, the invention includes methods for evaluating theefficacy of a treatment in a subject. The methods include determining afirst (e.g., baseline) level of circulating ST2 and/or IL-33 in asubject; comparing the first level of circulating ST2 and/or IL-33 inthe subject to a predetermined reference level; selecting the subject ifthe first level of ST2 is above the predetermined reference level;administering a treatment to the subject; determining a second level ofcirculating ST2 and/or IL-33 in a subject; and comparing the first andsecond levels of circulating ST2 and/or IL-33. A difference between thefirst and second levels of circulating ST2 and/or IL-33 indicates theefficacy of the treatment in the subject. For example, a second level ofcirculating ST2 and/or IL-33 that is lower than the first levelindicates that the treatment is effective.

As used herein, a “sample” includes any bodily fluid or tissue, e.g.,one or more of blood, serum, plasma, urine, and body tissue. In certainembodiments, a sample is a serum, plasma, or blood sample.

An antibody that “binds specifically to” an antigen, bindspreferentially to the antigen in a sample containing other proteins.

The methods and kits described herein have a number of advantages. Forexample, the methods can be used to determine whether a patient shouldbe admitted or held as an inpatient for further assessment, regardlessof whether a definitive diagnosis has been made. For example, themethods can be used for risk stratification of a given subject, e.g., tomake decisions regarding the level of aggressiveness of treatment thatis appropriate for the subject, based on their ST2 levels. Bettertreatment decisions can lead to reduced morbidity and mortality, andbetter allocation of scarce health care resources. The methods describedherein can be used to make general assessments as to whether a patientshould be further tested to determine a specific diagnosis. The methodsdescribed herein can also be used for patient population riskstratification, e.g., to provide information about clinical performanceor expected response to a therapeutic intervention. The methodsdescribed herein can be used regardless of the underlying cause orultimate diagnosis, and therefore are not limited to specificindications.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph illustrating receiver operating characteristicanalysis for ST2 and death within one year. ST2 was useful for thispurpose, as indicated by the high area under the curve (AUC).

FIG. 2 is a bar graph illustrating the crude rates of death acrossdeciles of ST2 in the ProBNP Investigation of Dyspnea in the EmergencyDepartment (PRIDE) study cohort. A clear threshold effect is noted atdecile 5, corresponding to an ST2 concentration of 0.23 ng/ml for theparticular assay.

FIGS. 3A and 3B are a pair of Kaplan-Meier hazard curves depicting therates of death from presentation to one year of follow up in patientswith dyspnea, stratified as a function of ST2 concentrations. Amongdyspneic patients with ST2 concentrations of ≥0.20 ng/ml, a high rate ofmortality was noted within days of presentation, and extending to a fullyear from presentation. The rates of death were similar among those with(3A) and without (3B) acute heart failure (all Log-rank p values<0.001).

FIG. 4 is a bar graph illustrating mortality rates as a function ofmarker concentrations for NT-proBNP and ST-2.

FIG. 5 is receiver operating curve (ROC) of specificity versussensitivity, for change in ST2 (light grey line) and change in BNP (darkline).

FIG. 6 is a combination bar and line graph. The bars illustrate thepercent mortality in populations with the indicated levels of BUN andST2 ratios. The line indicates the number of patients that are in eachcategory.

FIG. 7A is a line graph of average ST2 values for survivors (light greysquares) and non-survivors (dark diamonds) on each day ofhospitalization.

FIGS. 7B-7D are whisker box plots bracketing the 25th and 75thpercentiles of ST2 levels (7B), BNP concentration (7C), and NT-proBNP(7D), plotted against time (over 6 days of hospitalization);S=survivors, D=decedents.

FIG. 8 is a line graph of ratios of ST2 values for survivors (light greysquares) and non-survivors (dark diamonds) as compared to baseline(around admission) on each day of hospitalization.

FIG. 9 is a ROC for ST2 ability to predict death by one year in PRIDEsubjects with a pulmonary diagnosis. Area under ROC=0.73; 95%CI=0.62-0.83; P<0.0001; Optimal cut: 0.20 ng/ml; 88% sensitive, 52%specific; PPV=22%; NPV=96%.

FIG. 10 is a box graph illustrating the correlation between ST2concentrations and risk of death within one year in PRIDE subjects witha pulmonary diagnosis.

FIG. 11 is a line graph illustrating the mortality rate as a function ofST2 concentration in PRIDE subjects with a pulmonary diagnosis. P<0.001.

FIGS. 12A-B are box graphs illustrating mean Glomerular Filtration Rate(GFR, 12A) and ST2 levels (12B) in a population of 133 subjects withmoderate to severe renal insufficiency.

FIG. 13 is a bar graph illustrating the distribution of ST2 levels inthe population described in Example 8.

DETAILED DESCRIPTION

Clinical evaluation of patients, particularly patients with non-specificsymptoms such as dyspnea or chest pain, is often challenging. Theresults described herein provide evidence that ST2 is useful in theprognostic evaluation of patients, regardless of the underlying cause oftheir disease. ST2 is a powerful indicator of severe disease andimminent death, as demonstrated herein in several completely differentpopulations with completely different symptoms (see Examples 1-6).

Predicting Mortality

Elevated concentrations of ST2 are markedly prognostic for death withinone year, with a dramatic divergence in survival curves for those withelevated ST2 soon after presentation, regardless of the underlyingdiagnosis. As one example, there is a dramatic relationship betweenelevations of ST2 and the risk for mortality within one year followingpresentation with dyspnea. The relationship between ST2 and death indyspneic patients was independent of diagnosis, and superseded all otherbiomarker predictors of mortality in this setting, including othermarkers of inflammation, myonecrosis, renal dysfunction, and mostnotably NT-proBNP, a marker recently described as having value forpredicting death in this population (Januzzi et al., Arch. Intern. Med.2006; 166(3):315-20). Indeed, most of the mortality in the study wasconcentrated among subjects with elevated ST2 levels at presentation;however, the combination of an elevated ST2 and NT-proBNP was associatedwith the highest rates of death within one year.

Such a multi-marker approach for risk stratification has been proposedfor patients with acute coronary syndromes (Sabatine et al., Circulation2002; 105(15):1760-3), but no such strategy has yet been proposed forthe evaluation for the patient with non-specific symptoms such asundifferentiated dyspnea or general complaint of chest pain.

Determining Severity of Disease

Elevated concentrations of ST2 are correlated with the presence ofsevere disease in a subject, regardless of the underlying cause of thedisease. As one example, in a population of patients presenting withchest pain, the highest levels of disease were associated with severedisease including chronic obstructive pulmonary disease (COPD),lymphoma, sepsis, alcohol abuse, and pulmonary embolism (see Example 5).

Therefore, for undiagnosed subjects, the methods described herein can beused to determine how aggressively a diagnosis should be sought; a highST2 level would indicate the presence of severe disease, and suggestthat the subject should be treated as a high-risk case. For subjectswith a known diagnosis, the methods described herein can be used to helpdetermine the severity of the underlying pathology; again, a higher ST2level is associated with more severe disease.

General Methodology

In general, the methods described herein include evaluating circulatinglevels (e.g., levels in blood, serum, plasma, urine, or body tissue) ofST2 and/or IL-33 in a subject, e.g., a mammal, e.g., a human. Theselevels provide information regarding the subject's likelihood ofexperiencing an adverse outcome, e.g., mortality, e.g., within aspecific time period, e.g., 30 days, 60 days, 90 days, 6 months, oneyear, two years, three years, or five years. These levels also provideinformation regarding the severity of disease in the subject. In someembodiments, the level of ST2 and/or IL-33 is determined once, e.g., atpresentation. In some embodiments, the level of ST2 and/or IL-33 isdetermined 2, 4, 6, 8, 12, 18, and/or 24 hours, and/or 1-7 days afterthe onset of symptoms. Where more than one level is determined, a ratioof ST2 can be calculated that quantifies whether and how much the levelof ST2 in the subject has increased or decreased.

In some embodiments, the level of ST2 and/or IL-33 is determined morethan once; in that case, the higher measurement, or the most recentmeasurement, can be used. In embodiments where the level of ST2 and/orIL-33 is determined more that once, the highest level can be used, orthe difference between the levels (i.e., the magnitude and direction ofthe difference) can be determined and used. Thus, a ratio of ST2 can bedetermined that represents the change (e.g., the magnitude anddirection, e.g., increase or decrease) in ST2 levels over time, e.g.,over the course of a few days, e.g., 3 days or more, or over the courseof weeks or months; the ratio is indicative of the subject's risk leveland the presence of severe disease. Levels of ST2 and/or IL-33 can alsobe determined multiple times to evaluate a subject's response to atreatment. For example, a biomarker level of ST2 and/or IL-33 takenafter administration of a treatment, e.g., one or more doses or roundsof a treatment, can be compared to levels of ST2 and/or IL-33 before thetreatment was initiated. The difference between the ST2 levels wouldindicate whether the treatment was effective; e.g., a reduction in ST2levels would indicate that the treatment was effective. The differencebetween the ST2 levels can also be used to monitor a subject'scondition, e.g., to determine if the subject is improving, e.g.,improving enough to be discharged from a hospital, to be treated lessaggressively, or to be followed up at greater time intervals.

Evaluating circulating levels of ST2 and/or IL-33 in a subject typicallyincludes obtaining a biological sample, e.g., serum, plasma or blood,from the subject. Levels of ST2 and/or IL-33 in the sample can bedetermined by measuring levels of polypeptide in the sample, usingmethods known in the art and/or described herein, e.g., immunoassayssuch as enzyme-linked immunosorbent assays (ELISA). Alternatively,levels of ST2 and/or IL-33 mRNA can be measured, again using methodsknown in the art and/or described herein, e.g., by quantitative PCR orNorthern blotting analysis.

Once a level or ratio of ST2 and/or IL-33 has been determined, the levelor ratio can be compared to a reference level or ratio. In someembodiments, e.g., where the level of ST2 is determined using an ELISA,e.g., as described in Example 1, the reference level will represent athreshold level, above which the subject has an increased risk of death,and/or has a severe disease. The reference level chosen may depend onthe methodology used to measure the levels of ST2. For example, in someembodiments, where circulating levels of soluble ST2 are determinedusing an immunoassay, e.g., as described herein, the reference level isabout 0.20, 0.23, or 0.29 ng/ml of serum, and a level of ST2 above thatreference level indicates that the subject has an increased risk ofdeath, and/or has a severe disease.

Where a ratio has been determined, e.g., using a first and secondmeasurement of ST2, the reference ratio will represent an amount anddirection of change that indicates whether the subject has an increasedrisk of death and/or has a severe disease. As one example, an ST2 ratiocan be calculated based on a first measurement, e.g., a baselinemeasurement taken when a subject presents for treatment, e.g., to an ED,and a second measurement, e.g., a measurement taken about three to fourdays later. If the ratio of first and second ST2 levels over time isabout 0.85 or higher, i.e., the ST2 levels have decreased less thanabout 15% (or have stayed the same or increased), then the subject has avery high risk of imminent death. Ratios below about 0.85 (where the ST2levels have decreased more than about 15%) indicate that the subject hasa lower risk of imminent death.

This information allows a treating physician to make more accuratetreatment decisions; for example, when the results of the determinationindicate that the subject has a level equal to or above a referencelevel, e.g., above about 0.20 ng/ml, 0.23 ng/ml, or 0.29 ng/ml of serum,or a ratio above a reference ratio, the subject may be admitted to thehospital as an inpatient, e.g., in an acute or critical care department.In some embodiments, comparison of ST2 to a reference level or ratio canbe used to determine a subject's prognosis. For example, when the levelof ST2 is measured using an ELISA, e.g., as described herein in Example1, the reference level can be used to determine prognosis as follows: anST2<about 0.20 ng/ml or 0.23 ng/ml indicates that the subject has a goodprognosis, e.g., is likely to recover; an ST2 of from about 0.20 ng/mlor 0.23 ng/ml to about 0.7 ng/ml indicates that the subject has a poorprognosis, e.g., is less likely to recover. Finally, an ST2 of greaterthan about 0.7 ng/ml indicates a very poor prognosis, e.g., the subjectis not likely to recover. As another example, a ratio of first andsecond ST2 levels over time above about 0.85 indicates a poor prognosis,while a ratio of about 0.85 of below indicates a good prognosis.

Additional testing may be performed, to determine the subject's actualcondition. More aggressive treatment may be administered either beforeor after additional testing. For example, in the case of a suspected MIthe subject may be sent for more extensive imaging studies and/orcardiac catheterization.

In some embodiments, both levels of ST2 and IL-33 are determined, andthe information from the comparison of both biomarkers with theirrespective reference levels provides cumulative information regarding anincreased risk of death, and/or presence of a severe disease in thesubject. In some embodiments, the ratio of ST2 to IL-33 may bedetermined, and the ratio compared to a reference ratio that representsa threshold ratio above which the subject has an increased risk ofdeath, and/or has a severe disease. In some embodiments, the presence ofIL-33/ST2 complexes is detected, and the level of such complexes isindicative of risk of death and/or the presence of severe disease.

In some embodiments, the methods include the use of additionaldiagnostic methods to identify underlying pathology. Any diagnosticmethods known in the art can be used, and one of skill in the art willbe able to select diagnostic methods that are appropriate for thesubject's symptoms. In some embodiments, the methods described hereininclude other diagnostic methods in addition to or as an alternative tothe measurement of other biomarkers, e.g., physical measurements of lungfunction or cardiac function as are known in the art.

For example, the methods described herein include measuring levels ofST2 and/or IL-33 and one or more additional biomarkers that aid in thesubject's diagnosis. As one example, for a subject who has chest pain ordyspnea, biomarkers indicative of cardiac disease can be measured, e.g.,cardiac troponin (cTn), e.g., cTnl, BNP, and/or ANP; alternatively or inaddition, biomarkers of pulmonary disease can be measured, e.g.,D-dimers for pulmonary embolism. Thus, in subjects presenting withsymptoms that include MI in their differential diagnoses, the methodscan include measuring levels of, cTnl, BNP or NTproBNP or proBNP inaddition to ST2 and/or IL-33, to determine whether the subject is havingan MI. In subjects presenting with symptoms that include heart failure(HF) in their differential diagnoses, the methods can include measuringlevels of BNP or NTproBNP or proBNP in addition to ST2 and/or IL-33, todetermine whether the subject is having HF. In subjects presenting withsymptoms that include COPD in their differential diagnoses, the methodscan include measuring lung function in addition to levels of ST2 and/orIL-33, to determine whether the subject has COPD. One of skill in theart will appreciate that there are a number of additional diagnosticmethods that can be applied, depending on the situation and thesubject's condition. In some embodiments, the methods include measuringlevels of BUN, and the presence of elevated BUN and elevated ST2 placesthe subject in the highest risk category.

ST2

The ST2 gene is a member of the interleukin-1 receptor family, whoseprotein product exists both as a trans-membrane form, as well as asoluble receptor that is detectable in serum (Kieser et al., FEBS Lett.372(2-3):189-93 (1995); Kumar et al., J. Biol. Chem. 270(46):27905-13(1995); Yanagisawa et al., FEBS Lett. 302(1):51-3 (1992); Kuroiwa etal., Hybridoma 19(2):151-9 (2000)). ST2 was recently described to bemarkedly up-regulated in an experimental model of heart failure(Weinberg et al., Circulation 106(23):2961-6 (2002)), and preliminaryresults suggest that ST2 concentrations may be elevated in those withchronic severe HF (Weinberg et al., Circulation 107(5):721-6 (2003)) aswell as in those with acute myocardial infarction (MI) (Shimpo et al.,Circulation 109(18):2186-90 (2004)).

The trans-membrane form of ST2 is thought to play a role in modulatingresponses of T helper type 2 cells (Lohning et al., Proc. Natl. Acad.Sci. U.S.A 95(12):6930-5 (1998); Schmitz et al., Immunity 23(5):479-90(2005)), and may play a role in development of tolerance in states ofsevere or chronic inflammation (Brint et al., Nat. Immunol. 5(4):373-9(2004)), while the soluble form of ST2 is up-regulated in growthstimulated fibroblasts (Yanagisawa et al., 1992, supra). Experimentaldata suggest that the ST2 gene is markedly up-regulated in states ofmyocyte stretch (Weinberg et al., 2002, supra) in a manner analogous tothe induction of the BNP gene (Bruneau et al., Cardiovasc. Res.28(10):1519-25 (1994)).

Tominaga, FEBS Lett. 258:301-304 (1989), isolated murine genes that werespecifically expressed by growth stimulation in BALB/c-3T3 cells; theytermed one of these genes St2 (for Growth Stimulation-Expressed Gene 2).The St2 gene encodes two protein products: ST2, which is a solublesecreted form; and ST2L, a transmembrane receptor form that is verysimilar to the interleukin-1 receptors. The HUGO Nomenclature Committeedesignated the human homolog, the cloning of which was described inTominaga et al., Biochim. Biophys. Acta. 1171:215-218 (1992), asInterleukin 1 Receptor-Like 1 (IL1RL1). The two terms are usedinterchangeably herein.

The mRNA sequence of the shorter, soluble isoform of human ST2 can befound at GenBank Acc. No. NM_003856.2, and the polypeptide sequence isat GenBank Acc. No. NP_003847.2; the mRNA sequence for the longer formof human ST2 is at GenBank Acc. No. NM_016232.4; the polypeptidesequence is at GenBank Acc. No. NP_057316.3. Additional information isavailable in the public databases at GeneID: 9173, MIM ID #601203, andUniGene No. Hs.66. In general, in the methods described herein, thesoluble form of ST2 polypeptide is measured.

Methods for detecting and measuring ST2 are known in the art, e.g., asdescribed in U.S. Pat. Pub. Nos. 2003/0124624, 2004/0048286 and2005/0130136, the entire contents of which are incorporated herein byreference. Kits for measuring ST2 polypeptide are also commerciallyavailable, e.g., the ST2 ELISA Kit manufactured by Medical & BiologicalLaboratories Co., Ltd. (MBL International Corp., Woburn, Mass.), no.7638. In addition, devices for measuring ST2 and other biomarkers aredescribed in U.S. Pat. Pub. No. 2005/0250156.

In some embodiments, the methods include determining the identity of thenucleotide sequence at RefSNP ID: rs1041973.

IL-33

IL-33 was recently identified as the ligand for ST2, and the presence ofincreased levels of IL-33 in various inflammatory disorders has beendescribed (see Schmitz et al., Immunity 23(5):479-90 (2005); U.S. Pat.Pub. No. 2005/0203046). In the methods described herein, IL-33 can bemeasured instead of or in addition to ST2. The ratio of ST2 to IL-33 canalso be determined.

The nucleic acid sequence of IL-33 can be found at GenBank Acc. No.NM_033439.2, and the polypeptide sequence is at GenBank Acc. No.NP_254274.1. Additional information is available in the public databasesat GeneID: 90865, MIM ID # *608678, and UniGene No. Hs.348390. IL-33 isalso known as Chromosome 9 Open Reading Frame 26 (C9ORF26); NuclearFactor from High Endothelial Venules (NFHEV); and Interleukin 33. Seealso Baekkevold et al., Am. J. Path. 163: 69-79 (2003).

Methods for measuring levels of IL-33 are known in the art, see, e.g.,Schmitz et al., Immunity. 23(5):479-90 (2005), and U.S. Pat. Pub. No.2005/0203046.

Other Biomarkers

The methods described herein can also include measuring levels of otherbiomarkers in addition to ST2 and/or IL-33. Suitable biomarkers includeproBNP, NT-proBNP, BNP, NT-proANP, proANP, ANP, troponin, CRP, IL-6,D-dimers, BUN, liver function enzymes, albumin, measures of renalfunction, e.g., creatinine, creatinine clearance rate, or glomerularfiltration rate, and/or bacterial endotoxin. Methods for measuring thesebiomarkers are known in the art, see, e.g., U.S. Pat. Pub. Nos.2004/0048286 and 2005/0130136 to Lee et al.; Dhalla et al., Mol. Cell.Biochem. 87:85-92 (1989); Moe et al., Am. Heart. J. 139:587-95 (2000);Januzzi et al., Eur. Heart J. 27(3):330-7 (2006); Maisel et al., J. Am.Coll. Cardiol. 44(6):1328-33 (2004); and Maisel et al., N. Engl. J. Med.347(3):161-7 (2002), the entire contents of which are incorporatedherein by reference. Liver function enzymes include alanine transaminase(ALT); aspartate transaminase (AST); alkaline phosphatase (ALP); andtotal bilirubin (TBIL).

In these embodiments, levels of ST2 and/or IL-33 and one or moreadditional biomarkers are determined, and the information from thecomparison of the biomarkers with their respective reference levelsprovides additional information regarding the subject's risk of deathand/or the presence of a severe disease in the subject, which mayprovide more accurate and specific information regarding the subject'srisk. The levels can then be compared to a reference ratio thatrepresents a threshold ratio above which the subject has an increasedrisk of death, and/or has a severe disease.

As one example, the methods can include determining levels of NT-proBNPand ST2. The levels indicate the subject's risk of death, e.g., as shownin Table 1A.

TABLE 1A Risk of Death Based on Circulating Levels of NT-proBNP and ST2ST2 < 0.20 ng/ml ST2 ≥ 0.20 ng/ml NT-proBNP < 986 pg/ml Lowest RiskMedium Risk NT-proBNP ≥ 986 pg/ml Medium Risk Highest Risk

As shown in Table 1A, the lowest risk of death, e.g., no greater risk ofdeath than in normal patients or healthy individuals, occurs when bothST2 and NT-proBNP levels are low, and the highest risk of death, i.e., astatistically significantly increased risk, e.g., greater than 20%increased risk of death, e.g., a greater than 30, 40, or 50% higher riskthan a normal patient or healthy individual, occurs when both ST2 andNT-proBNP levels are high.

As another example, the methods can include determining levels of ST2and BUN. The levels indicate the subject's risk of death, e.g., as shownin Table 1B.

TABLE 1B Risk of Death Based on Circulating Levels of BUN and ST2 ST2 <0.20 ng/ml ST2 ≥ 0.20 ng/ml BUN < 40 mg/dL Lowest Risk Medium Risk BUN ≥40 mg/dL Medium Risk Highest Risk

As shown in Table 1B, the lowest risk of death, e.g., no greater risk ofdeath than in normal patients or healthy individuals, occurs when bothST2 and BUN levels are low, and the highest risk of death, e.g., astatistically significantly increased risk of death, e.g., a riskgreater than 30, 40, or 50% higher risk than a normal patient or healthyindividual, occurs when both ST2 and BUN levels are high.

Selecting a Treatment—Aggressive Vs. Conservative

Once it has been determined that a subject has a circulating level ofST2 and/or IL-33 above a predetermined reference level, the informationcan be used in a variety of ways. For example, if the subject haselevated ST2 levels, e.g., as compared to a reference level, a decisionto treat aggressively can be made, and the subject can be, e.g.,admitted to a hospital for treatment as an inpatient, e.g., in an acuteor critical care department. Portable test kits could allow emergencymedical personnel to evaluate a subject in the field, to determinewhether they should be transported to the ED. Triage decisions, e.g., inan ED or other clinical setting, can also be made based on informationprovided by a method described herein. Those patients with high ST2and/or IL-33 levels can be prioritized over those with lower ST2 orIL-33 levels.

The methods described herein also provide information regarding whethera subject is improving, e.g., responding to a treatment, e.g., whether ahospitalized subject has improved sufficiently to be discharged andfollowed on an outpatient basis. In general, these methods will includedetermining the levels of ST2 and/or IL-33 in the subject multipletimes. A decrease in ST2 and/or IL-33 levels over time indicates thatthe subject is likely to be improving. The most recent levels of ST2and/or IL-33 can also be compared to a threshold, as described herein,to determine whether the subject has improved sufficiently to bedischarged.

The subject may also be considered for inclusion in a clinical trial,e.g., of a treatment that carries a relatively high risk. The subjectcan be treated with a regimen that carries a relatively higher risk thanwould be considered appropriate for someone who had a lower risk ofimminent mortality, e.g., mortality within 30 days or within 1 year ofpresentation.

Beyond the clinical setting, information regarding a subject's ST2and/or IL-33 can be used in other ways, e.g., for payment decisions bythird party payors, or for setting medical or life insurance premiums byinsurance providers. For example, a high level of ST2 and/or IL-33,e.g., a level above a predetermined threshold level, may be used todecide to increase insurance premiums for the subject.

Patient Populations

The methods described herein are useful in a wide variety of clinicalcontexts. For example, the methods can be used for general populationscreening, including screening by doctors, e.g., in hospitals andoutpatient clinics, as well as the ED. As one example, levels of ST2and/or IL-33 can be determined at any time, and if ST2 and/or IL-33 iselevated, the physician can act appropriately.

Although the methods described herein can be used for any subject, atany time, they are particularly useful for those subjects for whom adiagnosis, or the severity of a condition, is difficult to determine.For example, such subjects may present with non-specific symptoms, e.g.,symptoms that do not indicate a specific diagnosis. Non-specificsymptoms include, but are not limited to, chest pain or discomfort,shortness of breath, nausea, vomiting, eructation, sweating,palpitations, lightheadedness, fatigue, and fainting. Each symptom canhave varied etiology.

Chest Pain

Chest pain is the chief complaint in about 1 to 2 percent of outpatientvisits, and although the cause is often noncardiac, heart diseaseremains the leading cause of death in the United States. Therefore,distinguishing between serious and benign causes of chest pain iscrucial. The methods described herein are useful in making thisdetermination.

A subject presenting to the ED with chest pain may have esophageal pain,an ulcer, acute lung problems such as pulmonary embolus (PE)(potentially fatal), rupturing or dissecting aneurysm (highly lethal),gall bladder attack, pericarditis (inflammation of the sack around theheart), angina pectoris (cardiac pain without damage), or an MI(potentially fatal). A precise diagnosis may be difficult to makeimmediately, but the decision whether to admit the subject or to treatthem conservatively should generally be made immediately. If the methodsdescribed herein indicate that the subject has an increased risk of anadverse clinical outcome, e.g., imminent mortality or severe disease,then the decision can be made to treat the subject aggressively, topotentially prevent the adverse outcome.

Additional information about treatment and diagnosis of chest pain maybe found, e.g., in Cayley, Am. Fam. Phys. 72(10):2012-2028 (2005).

Dyspnea

Dyspnea, or shortness of breath (also defined as abnormal oruncomfortable breathing), is a common symptom of subjects onpresentation to the ED. The differential diagnosis for dyspnea includesfour general categories: (1) cardiac, (2) pulmonary, (3) mixed cardiacor pulmonary, and (4) noncardiac or nonpulmonary.

Cardiac causes of dyspnea include right, left, or biventricularcongestive heart failure with resultant systolic dysfunction, coronaryartery disease, recent or remote myocardial infarction, cardiomyopathy,valvular dysfunction, left ventricular hypertrophy with resultantdiastolic dysfunction, asymmetric septal hypertrophy, pericarditis, andarrhythmias.

Pulmonary causes include obstructive (e.g., chronic obstructivepulmonary disease (COPD) and asthma) and restrictive processes (e.g.,extrapulmonary causes such as obesity, spine or chest wall deformities,and intrinsic pulmonary pathology such as interstitial fibrosis,pneumoconiosis, granulomatous disease or collagen vascular disease).

Mixed cardiac and pulmonary disorders include COPD with pulmonaryhypertension and cor pulmonale, deconditioning, pulmonary emboli, andtrauma.

Noncardiac or nonpulmonary disorders include metabolic conditions suchas anemia, diabetic ketoacidosis and other, less common causes ofmetabolic acidosis, pain in the chest wall or elsewhere in the body, andneuromuscular disorders such as multiple sclerosis and musculardystrophy. Obstructive rhinolaryngeal problems include nasal obstructiondue to polyps or septal deviation, enlarged tonsils, and supraglottic orsubglottic airway stricture.

Dyspnea can also present as a somatic manifestation of psychiatricdisorders, e.g., an anxiety disorder, with resultant hyperventilation.

Additional information regarding the evaluation and treatment of dyspneacan be found, e.g., in Morgan and Hodge, Am. Fam. Phys. 57(4):711-718(1998).

Special Populations

Certain populations of subjects may benefit particularly from themethods described herein. These subjects include people for whom BNP orNT-proBNP is less useful, such as in those with impaired renal function(Anwaruddin et al., J. Am. Coll. Cardiol. 47(1):91-7 (2006); McCulloughet al., Am. J. Kidney Dis. 41(3):571-9 (2003)), or in those who areoverweight (Body Mass Index (BMI) of 25-29) or obese (BMI≥30) (Krauseret al., Am. Heart J. 149(4):744-50 (2005); McCord et al., Arch. Intern.Med. 164(20):2247-52 (2004)). It is known and accepted in the field thatpatients with a high BMI usually have levels of natriuretic peptide thatare lower than expected relative to a normal body mass patient for thesame level of disease; the exact mechanism for this phenomenon is notknown. It has been shown that circulating levels of ST2 are notinfluenced by BMI, therefore, the determination of ST2 levels is moreuseful than natriuretic peptide levels in subjects with high BMI. Thus,the methods described herein can include determining a subject's BMI,and if the subject is overweight or obese, selecting the patient fordetermination of ST2 and/or IL-33 levels, as described herein.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Sandwich ELISA Assay

This example uses the ST2 ELISA Kit manufactured by Medical & BiologicalLaboratories Co., Ltd. (MBL International Corp., Woburn, Mass.), no.7638. This kit is a sandwich ELISA assay utilizing monoclonal antibodiesfor both capture and detection. This procedure is intended to analyze afull plate of samples assayed in replicates at a 1:3 dilution factor andclosely follows the manufacturers' protocol. Kits should be stored at 4°C. until use. The procedure described in this example is optimized forhuman serum or plasma collected in citrate or EDTA anticoagulant tubes.Plasma collected in heparin anticoagulant tubes should not be used inthis assay as heparin binds ST2 and inhibits measurement by this ELISAprotocol. Plasma or serum samples may be used fresh or stored frozen.This assay is not adversely effected by up to 3 freeze and thaw cyclesof plasma samples.

Reagents should be prepared fresh from a new kit immediately beforeperforming the assays. Allow the kit to equilibrate to room temperatureprior to use. Reagents not explicitly discussed below are provided bythe manufacturer ready to use.

-   -   1. Wash solution—wash solution is provided by the manufacturer        as a 10× concentrate solution. To make 1 liter of wash solution        dilute 100 ml of the 10× concentrate provided with 900 ml of        distilled water.    -   2. Detector solution—the detector solution is prepared by        diluting the detector concentrate 1:101 with the detector        diluent. For a full 96 well plate of samples 10 ml of detector        solution is required. To prepare 10 ml of detector solution use        a pipette to transfer 10 ml of the blue colored detector diluent        to a 15 ml orange top polypropylene tube. Ad 100 μl of the        detector concentrate to this volume of detector diluent.        -   a. NOTE: this reagent should be prepared during the first            assay incubation step.    -   3. Calibrator stock—reconstitute the calibrator protein by        dissolving the lyophilized protein in the amount of distilled        water defined by the manufacturer for this manufacturing lot to        yield a stock solution of 8 ng/ml. This volume specification is        included in the product insert.

Preparation of Standards and Samples:

-   -   All of the following should be prepared in labeled 1.5 ml        polypropylene tubes to be transferred to the assay plate with        the P200 pipetter.

Standards:

The standard curve is prepared by making 2 fold serial dilutions of the8 ng/ml stock solution.

-   -   1. Using a P1000 pipette transfer 250 μl of Assay Diluent to 8        1.5 ml polypropylene tubes labeled S1-S8.    -   2. Using the same P1000 pipette transfer 250 μl of the 8 ng/ml        Calibrator stock solution to tube 51. This tube is now 4 ng/ml        calibrator protein.        -   a. Mix thoroughly by gently pipetting 3 times being careful            not to create bubbles.    -   3. Using the same P1000 pipette and a fresh tip for each of the        following transfer 250 μl of the reagent in tube 51 to tube S2,        repeat the mixing.    -   4. Repeat step 3 for S2 to S3, S3 to S4, S4 to S5, S5 to S6 and        S6 to S7. S8 will be the reagent blank so do not transfer the        calibrant protein to this well.        -   a. Tubes S1-S6 and S8 will now have 250 μl of reagent and            tube S7 will have 450 μl

Samples:

The plate is set up so that each sample is analyzed as a 1:3 dilution induplicate.

-   -   1. Label a 1.5 ml polypropylene tube for each sample.    -   2. Using the P200 pipette transfer 160 μl of Assay Diluent to        each tube.    -   3. Using a P200 pipette transfer 80 μl of serum or plasma from        sample 1 to tube 1. Mix carefully by pipetting 3 times without        making bubbles.    -   4. Continue transferring samples to the sample tubes by        repeating step 2 for each sample.

Procedure:

-   -   1. Use the P200 pipette transfer the standards and diluted serum        samples quickly to the 96 well assay plate, as shown in Table 2.        -   a. Set the P200 pipette for 100 μl        -   b. Transfer 100 μl of the standard curve dilutions to each            of columns 1 & 2 in the assay plate        -   c. Transfer 100 μl of each of the serum samples to the assay            plate in exactly the same positions as shown in the plate            map below.    -   2. Cover the assay plate with the provided shield and incubate        at room temperature for 60 minutes.    -   3. Using the plate autowasher wash the plate 4 times.    -   4. Detector: using the 8 channel multichannel pipette transfer        100 μl of the detector solution to each well and incubate at        room temperature for 60 minutes.        -   a. NOTE: this reagent was to be prepared during the first            incubation step.        -   b. NOTE: use a disposable reagent vessel for this reagent            addition. ALWAYS use a fresh disposable reagent vessel for            each reagent. It is not necessary to change pipette tips            during this step.    -   5. Wash the plate as in step 3    -   6. Substrate: using the 8 channel multichannel pipette transfer        100 μl of the Substrate to each well and incubate at room        temperature for 30 minutes.        -   a. The Substrate reagent is provided ready to use by the            manufacturer.    -   7. Stop: at the completion of the Substrate incubation using the        8 channel multichannel pipette transfer 100 μl of the Stop        solution to each well.        -   a. The Stop Solution reagent is provided ready to use by the            manufacturer.    -   8. Read the plate at 450 nm with background correction at 620        nm.        -   a. The plate should be read within 30 minutes after stopping            the reaction.    -   9. Enter the absorbance readings in the provided spreadsheet for        analysis.

TABLE 2 Map of Exemplary 96 Well Assay Plate 1 2 3 4 5 6 7 8 9 10 11 12A 4.0 1 1 9 9 17 17 25 25 33 33 B 2.0 2 2 10 10 18 18 26 26 34 34 C 1.03 3 11 11 19 19 27 27 35 35 D 0.5 4 4 12 12 20 20 28 28 36 36 E 0.25 5 513 13 21 21 29 29 37 37 F 0.125 6 6 14 14 22 22 30 30 38 38 G 0.0625 7 715 15 23 23 31 31 39 39 H 0.0 8 8 16 16 24 24 32 32 40 40Table 2 is a map of an exemplary 96 well assay plate, with controlreactions in column 1, and each sample 1-40 analyzed in duplicate incolumns 3-12.

Example 2. Measurement of Soluble ST2 Concentrations for the Evaluationof Patients with Acute Dyspnea

In this example, the utility of ST2 measurement for evaluation ofdyspneic patients was assessed.

The subjects used in this Example took part in the ProBNP Investigationof Dyspnea in the Emergency Department (PRIDE) Study, a prospective,blinded study of 599 dyspneic subjects presenting to the ED of theMassachusetts General Hospital, and was performed for the purpose ofvalidation of the diagnostic and prognostic use of NT-proBNP testing.The results of the PRIDE study were recently reported (Januzzi et al.,Am. J. Cardiol. 95(8):948-54 (2005)).

The gold standard for the diagnosis of acute HF was based on theimpression of reviewing physicians, blinded to NT-proBNP values, who hadall available information from presentation through the 60-days offollow-up; for the few patients in whom a diagnosis was uncertain, thereviewers were instructed to utilize the guidelines as reported by theFramingham Heart Study (McKee et al., N. Engl. J. Med. 285(26):1441-6(1971)).

As reported, 209 subjects (35%) in the PRIDE study were adjudicated tohave dyspnea due to acute destabilized HF, of whom 17 had mild (ClassII) symptoms by the New York Heart Association (NYHA) classification, 80had moderate (Class III) symptoms, and 112 had severe (Class IVsymptoms). Of those without acute HF, the most common diagnoses wereexacerbation of obstructive airways disease (n=150; includesexacerbation of chronic obstructive pulmonary disease (n=120) and asthma(n=30) as well as acute pneumonia (n=64).

At the end of one year, the managing physician for each patient wascontacted to ascertain the vital status of the patient. As reported,follow-up at one year was complete in 597 subjects overall (Januzzi etal., Arch Intern Med 2006; 166(3):315-20).

NT-proBNP was measured in the PRIDE study using a commercially availableimmunoassay (ELECSYS® ProBNP assay, Roche Diagnostics, Indianapolis,Ind.), using established methodology. In the PRIDE study, the assay hadinter-run coefficients of variation of 0.9%. Blood collected at the timeof presentation was later analyzed for concentrations of ST2, using anenzyme-linked immunosorbent assay (Medical & Biological LaboratoriesCo., Ltd.), as described in Example 1 herein. This assay utilizesmonoclonal antibodies to human ST2 for both capture and detection, andhad a relative percent difference of 17.5% in the present analysis. Theplasma used for the present study had been previously subjected to asingle freeze-thaw cycle.

Comparisons Between Groups

Comparisons of clinical characteristics between patients were performedutilizing chi-square tests for categorical data and the Wilcoxonrank-sum test for continuous data. Comparisons of ST2 concentrationsbetween diagnostic, New York Heart Association (NYHA) symptom classesand outcome categories were performed using non-parametric testing.

Correlations

ST2 and NT-proBNP results were log-transformed to establish normaldistribution. Correlations between these log-transformed variables wereevaluated with the Spearman correlation coefficient. There was a modestcorrelation between concentrations of log-transformed ST2 andlog-NT-proBNP in all subjects (r=0.58, p<0.001), those without acute HF(r=0.47, p<0.001) and those with acute HF (r=0.40, p<0.001).

Cut-Point Analyses

Patient characteristics as a function of an ST2 concentration of aboveor below 0.20 ng/ml are detailed in Table 3, which demonstrates theexpected prevalence of factors consistent with a diagnosis of incidentHF.

TABLE 3 Characteristics of Study Subjects as a Function of ST2Concentrations ST2 ≥ 0.2 ng/ml ST2 < 0.2 ng/ml Characteristic (n = 320)(n = 279) P value Age (mean ± SD), years 67.7 ± 15.0 56.5 ± 17.5 <0.001Past medical history Prior cardiomyopathy 13% 7% 0.02 Prior congestiveheart failure 37% 12% <0.001 Arrhythmia 20% 13% 0.02 Hypertension 54%43% 0.009 Diabetes mellitus 35% 16% <0.001 Coronary artery disease 33%22% 0.005 Myocardial infarction 15% 11% NS Obstructive airway disease15% 8% 0.005 Symptoms/signs Paroxysmal nocturnal dyspnea 16% 8% 0.002Orthopnea 22% 12% 0.001 Lower extremity edema 26% 7% <0.001 Chest pain35% 52% <0.001 Dyspnea at rest 50% 24% <0.001 Medications atpresentation Beta blocker 44% 32% 0.002 Loop diuretic 41% 17% <0.001Digoxin 13% 8% 0.04 Angiotensin converting enzyme 25% 16% 0.009inhibitor Physical examination Body-mass index (Kg/m², mean ± SD) 28.0 ±7.0  28.5 ± 6.5  NS Pulse, beats per minute (mean ± SD) 91.7 ± 23.8 82.9± 20.6 <0.001 Jugular venous distension 13% 4% <0.001 Murmur 14% 8%0.009 Hepatojugular reflux 3% 0% 0.002 Lower extremity edema 34% 14%<0.001 Rales 35% 16% <0.001 Electrocardiographic findings Atrialfibrillation 16% 9% 0.009 Chest radiographic findings Interstitial edema25% 8% <0.001 Pleural effusion 28% 5% <0.001 Cephalization of vessels 2%0% 0.04 Laboratory findings Serum creatinine, mg/dl, mean ± SD 1.2 ± 0.50.98 ± 0.3  <0.001 Creatinine clearance, ml/min/1.73 m², 67.3 ± 32.085.2 ± 61.0 <0.001 mean ± SD Blood urea nitrogen, mg/dl, mean ± SD 25.5± 17.0 17.2 ± 10.0 <0.001 Troponin T, ng/dl, mean ± SD 0.063 ± 0.32 0.022 ± 0.16  0.04 NT-proBNP, pg/ml, median (IQR) 1800 (365-6745) 97(40-477) <0.001These results indicate that ST2 is not generally correlated with BMI,but is associated with a number of other indices including serumcreatinine levels and clearance, prior congestive heart failure, anddiabetes mellitus.

Example 3. Measurement of Soluble ST2 Concentrations for theDetermination of Risk of Death in Patients with Acute Dyspnea

Factors predictive of mortality within one year following presentationwith dyspnea were evaluated in the population described in Example 2.Candidate ST2 diagnostic cut points were evaluated with the use ofbootstrapping techniques using the STATA SWBOOT program; this wasfollowed by multivariable logistic regression analyses. Each of theestimation procedures was coded in programming language then subjectedto the STATA bootstrap prefix command for 10 bootstrap repeated randomsamples, followed by 100 replications for those variables selected inthe initial analyses. The bootstrap sample size was 593 (the size of theentire data set). Factors entered into the analysis included elementsfrom past and present medical history, symptoms and signs, medicationuse, as well as results of diagnostic studies including radiographicstudies, electrocardiography, hematology, and blood chemistries.Measures of renal function included serum creatinine results as well asestimated glomerular filtration rate (Levey et al., Ann. Intern. Med.130(6):461-70 (1999)). Following, candidate predictors of mortality(i.e., those with greater than 70 selections in bootstrap replications)were entered into multivariable logistic regression analyses, with acuteHF as the dependent variable. For each logistic regression, results wereentered in a single forward step with tail-probability to enter set atp=0.01 and to remove the effect from the regression at p=0.02, andgoodness-of-fit was evaluated using the Hosmer-Lemeshow test.

In mortality analyses including NT-proBNP, this variable was modeleddichotomously, with a threshold value of 986 pg/ml for predicting deathwithin one year, as identified previously (Januzzi et al., 2006, supra).After the SWBOOT run, the resulting validated candidate independentvariables were entered stepwise into a Cox Proportional Hazards model;the proportions for this model were checked and were found to beappropriate. Hazard ratios (HR) with 95% CI (confidence interval) weregenerated for each independent predictor of death by one year.

Kaplan-Meier survival curves were constructed to compare mortality rateswithin one year in groups divided as a function of ST2 concentrations(as well as diagnosis), using the log-rank test to compare thesignificance of the rates of mortality.

For all statistical analyses, either SPSS (Chicago, Ill., USA) or STATA(College Station, Tex.) software was used; all p values are two-sided,with composite results <0.05 considered significant.

Within one year, 93 subjects (15.7%) had died. The median concentrationsof ST2 were significantly higher among decedents (1.03 ng/ml, IQR(interquartile range)=0.38-2.43) than survivors (0.18 ng/ml,IQR=0.08-0.51; p<0.001). This pattern of higher ST2 concentrations indecedents remained when subjects were considered as a function of theabsence of acute HF (1.14 vs 0.13 ng/ml; p<0.001), as well as in thosewith acute HF (0.90 vs 0.45 ng/ml; p<0.001).

Receiver operating characteristic (ROC) curve analysis using Analyse-Itsoftware (Analyse-It, Ltd, Leeds, UK) was performed for ST2, using thegold standard diagnosis of HF or survival within one year as thereference standard, and area under the curve (AUC) was estimated;following, potential cut points for both diagnosis and prognosis wereidentified, and their sensitivity, specificity, as well as positive andnegative predictive values (PPV, NPV) were estimated. To furtherevaluate the utility of optimal ROC-optimal cut-points, patients werealso divided into deciles based on their ST2 concentrations, andevaluated for ST2 threshold effects on the frequency of mortality.

ROC analyses demonstrated an AUC of 0.80 (95% CI=0.75-0.84, p<0.001) forST2 and one year mortality (FIG. 1); the optimal cut point as identifiedby ROC was 0.29 ng/ml, which was 87% sensitive (95% CI=79-93%), 63%specific (95% CI=58-67%), and had a PPV of 30% and NPV of 96%. In decileanalyses, a threshold effect for mortality was noted at the ST2 medianof 0.20 ng/ml (FIG. 2). A graded relationship between ST2 concentrationsand the likelihood for death was also found, such that those subjectsbelow the median concentration of ST2 (n=236) enjoyed the lowest ratesof death (2%) compared to those in the highest two deciles (n=117) whodemonstrated a 42% rate of death within one year, a HR of 43.0 (95%CI=15.0-123.0, p<0.001).

In a final bootstrap model for prediction of death within one year(Table 4), an ST2 concentration ≥0.20 ng/ml was selected in 96 of 100replications and represented the strongest predictor of death within oneyear in breathless patients (HR=5.6, 95% CI=2.2-14.2; p<0.001). Notably,even with the inclusion of NT-proBNP into the models, an ST2≥0.20 ng/mlremained the most selected variable in bootstrap replications (86 of 100selections), and remained the strongest predictor of death within oneyear (HR=4.6, 95% CI=1.8-11.8; p=0.002).

TABLE 4 Identification of Independent Predictors of Death Within OneYear in Dyspneic Subjects Bootstrap Analysis Multivariable analysisNumber selected out of Hazard 95% confidence Variable 100 replicationsratio intervals P value Without NT-proBNP in the model Log-transformed99 2.7 1.6-4.4 <0.001 ST2 ST2 ≥ 0.20 ng/ml 96 5.6  2.2-14.2 <0.001Hemoglobin 73 0.91 0.85-0.98 0.008 Pleural effusion on 95 1.8 1.2-2.80.005 chest X-ray With NT-proBNP in the model ST2 ≥ 0.20 ng/ml 86 4.6 1.8-11.8 0.002 NT-proBNP ≥ 986 pg/ml 85 2.3 1.3-4.0 0.002 Cough 80 0.600.39-0.95 0.03 Pleural effusion on 80 1.6 1.0-2.4 0.05 chest X-rayLog-transformed 77 2.6 1.6-4.4 <0.001 ST2

The results of Table 4 are based on 100 bootstrap replications, followedby multivariable Cox Proportional Hazards analysis are shown in theabsence and presence of NT-proBNP results in the model. Only thosevariables selected >70 of 100 times in bootstrap replications are shown.

Kaplan-Meier hazard curves demonstrate that among subjects with ST2concentrations ≥0.20 ng/ml, rates of death rose rapidly from enrollment,and continued to rise within one year (FIG. 3A; Log rank p value<0.001). Similar relationships between ST2 values ≥0.20 ng/ml in thosewithout and with the diagnosis of acute HF at presentation (FIG. 3B;Log-rank p value for both <0.001).

Therefore, ST2 levels are an excellent predictor of mortality.

Example 4. Measurement of Soluble ST2 and NT-proBNP Concentrations forthe Determination of Risk of Death in Patients with Acute Dyspnea

As ST2 and NT-proBNP were both independent predictors of death withinone year, crude rates of death in subjects as a function of ST2 andNT-proBNP concentrations were examined using the methods describedabove. The percentage of subjects who died in each category are shown inTable 5.

TABLE 5 Mortality Rates as a Function of NT-proBNP and ST2Concentrations in the PRIDE Study ST2 < 0.20 ng/ml ST2 ≥ 0.20 ng/mlNT-proBNP < 986 pg/ml 1.7% 13.1% NT-proBNP ≥ 986 pg/ml 4.5% 37.0%

The majority of mortality among subjects in the PRIDE study occurred inthose with elevated levels of ST2 (equal to or above 0.20 ng/ml) andNT-proBNP (equal to or above 986 pg/ml) (Table 5 and FIG. 4).

Thus, the combination of NT-proBNP and ST2 levels is useful inpredicting risk of mortality.

Example 5. Elevated ST2 Concentrations in Patients in the Absence of MI

ST2 concentrations were determined as described in Example 1, above, ina population of 350 patients who presented to the ED with chest pain.Serum samples were obtained and ST2 measurements made at baseline, and90 and 180 minutes later for most patients. Also, for most patients, thebaseline sample was collected within 2 hours of onset of symptoms.

17 patients had final diagnosis of MI, and 5 of these had ST2≥0.23(0.25-0.65). Two of these patients were troponin negative. 11 patientshad very high ST2 levels (0.97-9.22), but none of these patients hadconfirmed final diagnosis of MI, though all had severe diseases,including COPD, lymphoma, sepsis, alcohol abuse, and pulmonary embolism.The diagnoses for these 11 patients are shown in Table 6; ST2 1 is thebaseline level, ST2 2 is 90 minutes later, and ST2 3 is at 180 minutes.

TABLE 6 Non-MI Patients with High ST2 Levels ST2 1 ST2 2 ST2 3 Patient(ng/ml) (ng/ml) (ng/ml) Final Diagnosis 811 1.43 1.62 1.63 COPD withheart failure following coronary artery bypass graft surgery andpulmonary hypertension 847 2.37 4.44 3.53 Pulmonary embolism 873 2.362.42 2.74 RAD 898 1.32 1.24 1.66 History of heart failure followingcoronary artery bypass surgery 920 6.03 9.22 Bacteremia sepsis 928 3.804.69 3.99 Hypertension and alcohol abuse 952 6.76 Alcohol abuse,gastritis and pulmonary hypertension 953 3.77 History of heart failurefollowing coronary artery bypass surgery 1055 1.42 1.28 1.13 URI 12130.97 1.19 1.07 Pulmonary embolism and pericarditis 1245 4.11 6.46Lymphoma and hypertension 1280 1.30 1.33 COPDThese results demonstrate that elevated ST2 is associated with severedisease, regardless of the underlying pathology.

Example 6. Serial Analysis of ST2 Concentrations in PatientsHospitalized with Acute Decompensated Heart Failure (ADHF)

ST2 concentrations were determined as described in Example 1, above, aswere BUN, NT-Pro-BNP, and BNP, in a population of 150 subjects who werediagnosed with and admitted for acute decompensated heart failure (ADHF)to the San Diego Veterans' Administration Hospital. Some patients had anew diagnosis of ADHF and other patients had an acute exacerbation ofexisting heart failure. Samples were taken on successive days in anumber of patients, though not every patient gave a sample every day.Length of stay (LOS) in the hospital for these patients ranged from 1 to24 days with an average of 5 days. Table 7 shows the characteristics ofthe population; as expected, given that the population was drawn fromthe San Diego Veteran's Hospital, the population was overwhelmingly maleand Caucasian.

TABLE 7 Frequency of Patient Characteristics Frequency Percent GenderMale 148 98.7 Female 2 1.3 Total 150 100 Ethnicity White 115 76.6 Black22 14.7 Hispanic 9 6 Asian 3 2 other 1 0.7 Total 150 100 Diagnosis CHFnew 14 9.3 CHF worse 110 73.3 Other 26 17.3 NYHA* Class II 9 6 Class III75 50 Class IV 66 44 Etiology Unknown 61 40.7 Ischemic 43 28.7Hypertensive 23 15.3 Other 9 6.1 Alcohol 5 3.3 Idiopathic 3 2 Valve 10.7 Drug 1 0.7 *NYHA = New York Heart Association Stages of HeartFailure: Class I: No limitation of physical activity. Ordinary physicalactivity does not cause undue fatigue, palpitation, or dyspnea(shortness of breath). Class II: Slight limitation of physical activity.Comfortable at rest, but ordinary physical activity results in fatigue,palpitation, or dyspnea. Class III: Marked limitation of physicalactivity. Comfortable at rest, but less than ordinary activity causesfatigue, palpitation, or dyspnea. Class IV: Unable to carry out anyphysical activity without discomfort. Symptoms of cardiac insufficiencyat rest. If any physical activity is undertaken, discomfort isincreased.

The population of patients was followed for at least 90 days, andadverse events were tabulated. A summary of events is shown in Table 8.

TABLE 8 Summary of Events Frequency Percent In hospital mortality 8 5.330 day readmission 13 8.7 30 day mortality CHF 10 6.7 30 day mortalityother 7 4.7 90 day readmission 26 17.3 90 day mortality CHF 26 17.3 90day mortality other 9 6.0 *90 day outcome frequency is cumulative of allevents that occurred at earlier time points

Receiver operating characteristics and area under the curve (AUC) weredetermined as described herein for the correlation of levels of BNP,NT-ProBNP, and ST2 at admission and at discharge with the adverse eventslisted in Table 8. The results are shown in Table 9. AUC for ST2 changefrom first to last=0.820.

TABLE 9 ROC SUMMARY (AUC) In Hospital 30 day 30 day 90 day 90 daymortality event mortality event mortality BNP Admit 0.735 0.536 0.6300.569 0.653 BNP NA 0.545 0.648 0.575 0.692 Discharge NT-proBNP 0.7900.638 0.785 0.624 0.763 Admit NT-proBNP NA 0.637 0.831 0.631 0.815Discharge ST2 admit 0.638 0.549 0.574 0.558 0.603 ST2 0.752 0.642 0.7600.657 0.772 Discharge Change in NA 0.573 0.793 0.604 0.809 ST2

In this small and extremely homogeneous population, the biomarkers thatmost accurately predicted the reported events included change in ST2 for30 and 90 day mortality, and NT-proBNP levels at discharge for both 30and 90-day mortality. As expected, the homogeneity of this cohortresulted in the AUC using the admission measurement for each biomarkerbeing lower than what was observed in the PRIDE cohort (described inexamples 2-3). The high number of patients without diagnosed heartfailure reported in the PRIDE cohort resulted in a high discriminatorypower of ST2 at the admission measurement. In this specialized cohort,where every patient already had a diagnosis of heart failure, the ST2level at admission is expected to be somewhat elevated; consequently,the predictive power for subsequent events is somewhat diminished ascompared to a heterogenous cohort that is more representative of thegeneral population.

A univariate model of predictors of 90 day mortality was analyzedincluding the same markers, as well as BUN and creatinine clearance,which are markers of renal function. The results are shown in Table 10.

TABLE 10 Univariate Predictors of 90-day Mortality Predictor Odds RatioP-Value Change in ST 2: ≥−0.02 vs. <−0.02 11.55 0.002 Admission ST 2:≥0.2 vs. <0.2 1.14 0.83 Discharge ST 2: ≥0.2 vs. <0.2 7.00 0.003Admission BNP: ≥400 vs. <400 1.91 0.35 Discharge BNP: ≥400 vs. <400 2.590.14 Admission NT-ProBNP: ≥5000 vs. <5000 3.96 0.09 Discharge NT-ProBNP:≥5000 vs. <5000 6.72 0.007 Admission BUN continuous 1.033 0.002Admission creatinine continuous 1.50 0.07

In this analysis, for the dichotomous variables (all but BUN andcreatinine) the odds ratio represents the increased odds of death within90 days for a positive test vs. a negative test. For the continuousvariables (BUN and creatinine) the odds ratio represents the increasedodds of death within 90 days for each 1 mg/dL increase. As shown inTable 10, change in ST2 provided the best indication of 90 daymortality, with an OR of 11.55 (p=0.002).

A multivariate model was also constructed that included BNP, NT-ProBNP,BUN and creatinine clearance levels with change in ST2. The results areshown in Table 11.

TABLE 11 Predictors of 90-day Mortality With Change in ST2 ChangeAdditional Additional in ST 2 Predictor Predictor in Odds Odds ModelModel Ratio P-Value Ratio P-Value R² Admission 12.18 0.002 2.04 0.340.246 BNP Discharge BNP 13.98 0.001 3.56 0.07 0.285 Admission NT- 14.490.001 5.48 0.044 0.312 ProBNP Discharge NT- 10.04 0.005 5.49 0.021 0.325ProBNP Admission 7.87 0.014 1.023 0.034 0.300 BUN Admission 10.08 0.0051.260 0.30 0.241 creatinine

When only change in ST2 (the difference between the ST2 levels) wasconsidered, the R² for the model was 0.223. As can be seen in Table 11,in each case change in ST2 was the stronger predictor due to its havingthe smaller p-value. Moreover, only admission NT-ProBNP, dischargeNT-ProBNP, and admission BUN provided additional prediction beyondchange in ST2 that achieved a statistical significance (p<0.05) (seebold R² values), while discharge BNP achieved marginal statisticalsignificance (p=0.07).

Next, the ROC was compared for change in BNP and change in ST2. As shownin FIG. 5, the AUC for change in BNP during hospitalization was 0.67,while the AUC for change in ST2 was 0.78, significantly higher,indicating that change in ST2 is a much more accurate predictor of90-day mortality than BNP.

The change in ST2 was analyzed in conjunction with BUN levels todetermine whether the two markers, when used together providedadditional predictive information. The results, shown in FIG. 6,demonstrate that BUN levels and changes in ST2 levels can be used tocategorize patients by risk: patients with low levels of BUN (less than40 mg/dL) and levels of ST2 that decreased by 15% or more had the lowestrisk levels; patients with higher BUN and levels of ST2 that decreasedby less than 15% (or increased or held steady) had the highest risklevels, while subjects with either high BUN and ST2 decreased by atleast 15%, or low BUN and ST2 decreased by 15% or more, had a mediumlevel of risk (which was still much increased over that of the lowestrisk category, see FIG. 6).

Changes in ST2 levels over time were evaluated on a lapsed-time basis todetermine an optimal length of time between first and second ST2measurements. The results, shown in Tables 11A and 11B, indicate that alapsed time of at least two days and within four days between the firstand second measurement of ST2 provides the best information regardinglikelihood of death within 90 days.

TABLE 11A Summary of ST2 Values over Time N average median 1 day 1300.97 0.78 2 days 99 1.00 0.60 3 days 66 1.42 0.42 4 days 44 1.87 0.57 5days 34 1.86 0.74

TABLE 11B Correlation of ST2 Values with Mortality within 90 DaysN >0.85 # dead % death N <0.85 # dead % death 1 day 54 7 13% 76 7 9% 2days 27 7 26% 72 4 6% 3 days 19 4 21% 47 2 4% 4 days 18 6 33% 26 1 4% 5days 14 6 43% 20 1 5%

In Table 11B, “N>0.85” indicates the number of people who had areduction of less than 15% in ST2 over the time period indicated (sothat the second level is 85% or more of the first level). After at leasttwo days, the percentage of people with a ratio of 0.85 or higher whodied was over 20% while the percentage of patients who died with a ratiobelow 0.85 initially decreased and remained low.

When average ST2 values over time were tracked in this population, aclear increase in correlation of risk of mortality within 90 days wasseen at three to four days after the initial measurement was made, asshown in Table 12A and FIGS. 7A and 7B. FIG. 7B, a whisker box plotbracketing the 25^(th) and 75^(th) percentiles, illustrates thatdistinction between survivors and decedents is clearly resolved with theST2 value. The ST2 results contrast with results generated by comparingeither BNP or NT-proBNP over these same days of treatment. Although themean values for each of these markers is distinct between survivors anddecedents the groups are not statistically distinct from each other, asshown in FIGS. 7C and 7D. Unlike ST2 the survivor and decedent groupsfor BNP and NT-proBNP overlap significantly between the 25^(th) and75^(th) percentiles. In this analysis ST2 is a more accurate marker thanBNP or NT-proBNP alone for predicting mortality as a function of changeover time during hospitalization.

When average ratios of ST2 were calculated, again, a significantdifference was seen; the results are shown in Table 12B and FIG. 8.

TABLE 12A Average ST2 Values Over Time Average ST2 value ST2-1 ST2-2ST2-3 ST2-4 ST2-5 ST2-6 non-survivor 0.33 0.38 0.44 1.14 0.89 0.71survivor 0.27 0.24 0.15 0.17 0.15 0.17

TABLE 12B Change in ST2 Over Time Average Ratio of ST2 1 day 2 days 3days 4 days 5 days non-survivor 1.04 2.44 9.57 8.43 6.46 survivor 0.960.82 0.60 0.63 0.67

It is also possible to use the ratio of ST2 along with the ratio ofNT-proBNP in a 2×2 matrix analysis to further refine the riskstratification of these patients, as shown in Table 12C.

TABLE 12C Change in ST2 and Change in NT-proBNP for Risk StratificationNT-proBNP and ST2 ratios NT <0.7, NT <0.7, NT >0.7, NT >0.7, ST2 <0.85ST2 >0.85 ST2 <0.85 ST2 >0.85 N 45 7 34 28 Mort 0 2 3 6 % Mort 0.0%28.6% 8.8% 21.4%

These results demonstrate that the change in ST2 over time is a powerfulpredictor of mortality, e.g., within 90 days, in both univariate ormultivariate predictive analysis; change in ST2 was shown to be thestrongest univariate predictor of patient outcome in this population,with optimal predictive value seen after 3 days of hospitalization.Combining change in ST2 data with a measure of renal function (e.g.,BUN) provided additional predictive value, as did including an NT-proBNPmeasurement with change in ST2.

Example 7. ST2 Concentrations for Risk Stratification in Patients withPulmonary Disease

Of the 599 subjects in the PRIDE population (described in Examples 2 and3), 209 had acute decompensated heart failure (ADHF). Of the remaining390 subjects, 236 had “pulmonary disease”; of those with available data,5 had uncomplicated bronchitis; 18 had pulmonary embolism (PE); 64 hadpneumonia; and 149 had COPD/asthma. Of the 149 with COPD/asthma, 69 hadasthma; 67 had emphysema; and 13 had chronic bronchitis.

To evaluate the usefulness of ST2 as a marker of risk in these patients,ST2 levels were assessed in each diagnostic category. Concentrations foreach diagnostic category are shown in Table 13.

TABLE 13 ST2 Concentrations by Diagnosis Category ST2 median 25th-75thPercentile Whole group 0.23 0.09-0.69 PE 0.16 0.08-1.4  Pneumonia 0.690.14-2.29 Bronchitis 0.19 0.11-0.41 Asthma/COPD 0.20 0.08-0.54 Asthma0.14 0.05-0.47 Emphysema 0.24 0.12-0.55 Chronic bronchitis 0.330.06-1.2 

ST2 concentrations were evaluated using ROC analysis for death at oneyear, and concentrations in those dead and alive at one year werecompared. Mortality rates as a function of ST2 concentrations to oneyear were analyzed. The results are shown in Table 14 and FIGS. 9-11.

TABLE 14 Mortality by Diagnosis PE PNA Acute bronchitis Asthma/COPD N 1864 5 149 N survivors 15 52 5 139 ST2 survivors 0.14 (0.07-0.36) 0.53(0.13-1.81) See above 0.17 (0.07-0.50) ST2 decedents 1.98 (1.2-3.83) 1.17 (.4-3.17)  n/a 0.31 (.2-1.1)  

These results demonstrate that, as shown in FIG. 9, ST2 concentrationsshowed excellent specificity and sensitivity (Area under ROC=0.73; 95%CI=0.62-0.83; P<0.0001). The optimal cut point was 0.20 ng/ml, with 88%sensitivity and 52% specificity (PPV=22%; NPV=96%). FIG. 10 furtherillustrates this point. The median ST2 concentrations in subjects whowere still alive at one year was 0.19 ng/ml (IQR 0.08-0.59, n=211);while median concentrations in subjects who had died within one yearwere 1.19 ng/ml (IQR 0.28-2.2, n=25). Finally, as shown in FIG. 11,mortality rate increased dramatically as a function of ST2concentration, using a threshold of 0.2 ng/ml.

Multivariate Cox Proportional Hazards analyses were used to identifyindependent predictors of death at one year; the results are shown intable 15.

TABLE 15 Independent Predictors of Death in Pulmonary DiseaseCharacteristic Hazard ratio 95% CI P value Age 1.01 0.98-1.04 0.71Gender 1.36 0.60-3.07 0.74 ST2 ≥ 0.20 ng/ml 6.14 1.80-21.0 0.004 Pleuraleffusion on 2.99 1.30-6.83 0.009 CXR Emphysema 0.29 0.13-0.65 0.003Spironolactone 13.7 3.60-52.0 <0.001

These data demonstrate that ST2 levels are an excellent predictor ofdeath within one year, regardless of the underlying pathology, insubjects presenting with dyspnea.

Example 8. ST2 Concentrations are Not Affected by Renal Insufficiency

The effect of renal impairment on ST2 concentrations was evaluated in apopulation of 135 patients with moderate to severe renal insufficiency.None of the patients were on dialysis, and none were previouslydiagnosed with CVD. All of the patients were evaluated using glomerularfiltration rate (GFR in mls/min) as determined by the Modification ofDiet in Renal Disease (MDRD) method as a measure of renal function.Echocardiography and coronary artery calcium (CAC) measurements werealso performed on each subject to detect latent CVD. Multiple biomarkerswere also evaluated.

The descriptive statistics for this cohort are shown in Table 16; themean GFR and ST2 are illustrated graphically in FIGS. 12A-B.

TABLE 16 Glomerular Filtration Rate (GFR) and ST2 Levels GFR ST2 levels(ng/ml) Mean 34.5 0.122 Median 34 0.107 Std Error 0.989 0.005 Std Dev.11.4 0.059 Coeff. Var. 33.3 48.346 Lower 95% CL 32.5 0.112 Upper 95% CL36.4 0.132 25th Percentile 27 0.090 75th Percentile 43 0.127 Minimum 90.068 Maximum 59 0.476 Count 135 135

In this cohort of patients with stable, chronic disease, only ten (8%)had ST2 levels above 0.2, the highest of which was 0.476 ng/ml. Thedistribution of ST2 values is shown in FIG. 13. This was as expected inthis population of subjects with chronic, managed renal insufficiency;one would not expect to see very high ST2 levels.

Pearson Correlation analysis was performed in this population todetermine whether there was a correlation between ST2 levels and GFR.The results are shown in Tables 17 and 18.

TABLE 17 Pearson Correlation Results - GFR and ST2 DescriptiveStatistics Variable Mean Std Dev. Std Err N GFR 34.5 11.5 0.989 135 ST2(ng/mL) 0.122 0.059 0.005 135 Correlation Matrix (R) GFR ST2 (ng/mL) GFR1.000 0.028 ST2 (ng/mL) 0.028 1.000 Correlation Significance (P) GFR ST2(ng/mL) GFR — 0.748 ST2 (ng/mL) 0.748 —

TABLE 18 Pearson Correlation Results - Creatinine Clearance and ST2Descriptive Statistics Variable Mean Std Dev. Std Err N Screening Cr2.175 0.859 0.081 113 ST2 (ng/mL) 0.122 0.058 0.006 113 CorrelationMatrix (R) Screening Cr ST2 (ng/mL) Screening Cr 1.000 −0.018 ST2(ng/mL) −0.018 1.000 Correlation Significance (P) Screening Cr ST2(ng/mL) Screening Cr — 0.851 ST2 (ng/mL) 0.851 —

These results demonstrate that, as was expected in this population ofsubjects with chronic, managed renal insufficiency, there is nocorrelation between ST2 levels and either GFR (p=0.75) or creatinineclearance (p=0.851) in this population. This indicates that renalinsufficiency, by itself, does not cause an elevation of ST2 levels.

The same analyses were carried out in a population of 139 subjects atthe San Diego Veteran's Administration Hospital. All of the subjects hadpreviously been diagnosed with acute decompensated heart failure (ADHF),and the mean ST2 level was about twice that seen in the population ofpatients with chronic renal insufficiency but no HF (see tables 17-18).There is an almost ubiquitous correlation between renal insufficiencyand heart failure, with an almost 80% confluence of patients with stageIII/IV HF also having impaired renal function (Fonarow and Heywood, Am.J. Med. (2006) 119(12A):S17-525. Thus, because ADHF is correlated withST2 levels, one would expect to see a correlation between renalinsufficiency (as measured by GFR) and ST2 levels. This was exactly whatwas seen, as shown in Tables 19 and 20.

TABLE 19 Pearson Correlation Results - GFR and ST2 in ADHF DescriptiveStatistics Variable Mean Std Dev. Std Err N GFR 59.1 25.3 2.143 139 ST2(ng/mL) 0.283 0.332 0.028 139 Correlation Matrix (R) GFR ST2 (ng/mL) GFR1.000 −0.062 ST2 (ng/mL) −0.062 1.000 Correlation Significance (P) GFRST2 (ng/mL) GFR — 0.470 ST2 (ng/mL) 0.470 —

TABLE 20 Pearson Correlation Results - GFR and ST2 Ratios in ADHFDescriptive Statistics Variable Mean Std Dev. Std Err N GFR 59.1 25.32.143 139 ST2 ratio 1.038 3.038 0.258 139 Correlation Matrix ® GFR ST2ratio GFR 1.000 −0.161 ST2 ratio −0.161 1.000 Correlation Significance(P) GFR ST2 ratio GFR — 0.058 ST2 ratio 0.058 —

These results demonstrate that, in subjects with ADHF, ST2 values,whether represented as a single level or a ratio, are correlated withmeasures of renal insufficiency, but are independent of the renalinsufficiency.

Example 9. Risk of Mortality Correlates with ST2 Concentrations

The amount of increased risk of mortality was evaluated in the subjectswho participated in the PRIDE study and the veterans in the populationdescribed in Example 8.

Both 0.2 and 0.7 ng/ml ST2 thresholds were evaluated across the entirepopulation (Table 21) and the just the ADHF subset (Table 22). Thebiggest difference between the two thresholds is that the PPV increasesand the NPV decreases for the 0.7 ng/ml threshold in both patient sets.

TABLE 21 % Mortality by ST2 level in PRIDE Cohort ST2 ≥0.7 ST2 <0.7 ST2≥0.2 ST2 <0.2 Total N 146 447 317 275 Mortality N 52 41 88 5 % Mortality35.6% 9.2% 27.8% 1.8%

The NPV for 0.2 ng/ml is 98.2% and for 0.7 ng/ml is 90.8% in thispopulation.

TABLE 22 % Mortality by ST2 level in Subjects with ADHF ST2 ≥0.7 ST2<0.7 ST2 ≥_0.2 ST2 <0.2 Total N 81 127 167 41 Mortality N 30 26 55 1 %Mortality 37.0% 20.5% 32.9% 2.4%

The NPV for 0.2 ng/ml is 97.6% and for 0.7 ng/ml is 79.5% in thiscohort.

For the ST2 ratio from the VET study the results are shown in Table 23.

TABLE 23 % Mortality in Subjects with Chronic Renal Failure ST2 ratio≥0.85 <0.85 N 35 79 Mortality 8 3 % Mort 22.9% 3.8%

The NPV for this measurement is 96.2%.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for determining the risk of death withinone year in a subject having dyspnea, and treating the subject, themethod comprising: (a) collecting a biological sample from said dyspneasubject at a first time point; (b) performing an assay to determine afirst level of soluble ST2 in said sample from said subject at the firsttime point; (c) collecting a biological sample from said dyspnea subjectat a second time point; (d) performing an assay to determine a secondlevel of soluble ST2 in the sample from the subject at the second timepoint; and (e) identifying a subject having a second level of solubleST2 that is equal or elevated as compared to the first level as havingan elevated risk of death within one year and (f) performing imagingstudies or cardiac catheterization on, the subject identified as havingan elevated risk of death within one year.
 2. The method of claim 1,wherein the biological sample in (a) and the biological sample in (b)each comprises serum, blood, or plasma.
 3. The method of claim 1,wherein the subject does not have a cardiovascular disorder.
 4. Themethod of claim 1, wherein the subject has a pulmonary disorder.
 5. Themethod of claim 1, further comprising: Collecting a biological sampleand performing an assay to determining a first level of one or moreadjunct marker(s) selected from the group consisting of troponin, brainnatriuretic peptide (BNP), proBNP, amino-terminal (NT) proBNP, atrialnatriuretic peptide (ANP), NT-proBNP, proANP, C-reactive protein, andD-dimers in the subject at the first time point; And collecting anotherbiological sample and performing an assay to determine a second level ofthe one or more adjunct biomarker(s) in the subject at the second timepoint.
 6. The method of claim 5, wherein the one or more adjunctbiomarker(s) is selected from the group consisting of: NT-proBNP andBNP.
 7. The method of claim 1, wherein the subject has a body mass index(BMI) of 25-29 or renal insufficiency.
 8. The method of claim 1, whereinthe subject has a BMI of ≥30.
 9. The method of claim 1, wherein the oneor more adjunct biomarker is NT-proBNP.
 10. The method of claim 1,further comprising obtaining the first and second biological samplesfrom the subject.
 11. The method of claim 1, wherein inpatient care ofthe subject identified as having an elevated risk of death within oneyear is continued or initiated, or imaging studies or cardiaccatheterization is performed on the subject identified as having anelevated risk of death within one year.
 12. The method of claim 11,wherein inpatient care of the subject identified as having an elevatedrisk of death within one year is initiated.
 13. The method of claim 11,wherein the inpatient care of the subject identified as having anelevated risk of death within one year is continued.
 14. The method ofclaim 1, wherein the subject identified as having a decreased risk ofdeath within one year is discharged from a hospital or provided care onan outpatient basis.
 15. The method of claim 14, wherein the subjectidentified as having a decreased risk of death within one year isdischarged from a hospital.
 16. The method of claim 14, wherein thesubject identified as having a decreased risk of death within one yearis provided care on an outpatient basis.
 17. The method of claim 1,wherein the assay to determine the first level of soluble ST2 in (a) andthe assay to determine the second level of soluble ST2 in (b) is animmunoassay.
 18. The method of claim 17, wherein the immunoassay is anenzyme-linked immunosorbent assay (ELISA).
 19. The method of claim 18,wherein the ELISA is a sandwich ELISA.
 20. The method of 17, wherein theimmunoassay utilizes a monoclonal antibody.